One of the major questions being pursued in biology today is: "How are specific genes turned on and off at the appropriate times in a developmental sequence?: The aquatic bacterium Caulobacter crescentus provides a simple system for the study of this question since differentiation occurs twice during the normal cell cycle. In each case, the differentiation is characterized by well-defined events at one pole of the cell. The ultimate goal of the research conducted in our laboratory is to elucidate the modes of regulation of the genes involved in this differentiation. The work contained in this proposal will involve in this differentiation. The work contained in this proposal will involve an analysis of the regulation of the gene expression in C. crescentus. We will examine both biosynthetic genes and those which are expressed at specific times in the cell cycle in order to determine how these genes are regulated. During the course of these studies, we will determine how the expression of these genes is tied to the cell cycle. We propose: 1) To characterize mutants with altered polar organelle development (pod) and to determine the role of pod genes in the regulation and timing of flagellar gene expression. 2) To determine the time in the cell cycle when cloned fla and pod genes are expressed using quantitative mRNA analyses, western analyses, and the fusion of reporter genes to fla promoters in order to monitor gene expression. 3) To use oligonucleotide mutagenesis to determine which nucleotides are essential for the biosynthetic and pod gene promoters and for a newly identified class of flagellar gene promoters. 4) To examine the mechanism by which the flbT repressor protein regulates flagellin gene expression by purifying the FlbT protein and analyzing its interaction with flagellin gene promoters.